Method for improving fluorescence image contrast

ABSTRACT

There is provided an improved method for enhancing fluorescence images of an object, such as a biological tissue, by selectively eliminating or reducing unwanted fluorescence from fluorophores other than the fluorophore of interest. The method is based on the measurement of the lifetime of fluorophores while preserving information related to the fluorescence intensity of the fluorophore of interest.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is the first application filed for the present invention.

TECHNICAL FIELD

The present invention relates to a method for reducing and/oreliminating unwanted fluorescence signals in optical images based onfluorescence lifetime of fluorophores.

BACKGROUND OF THE INVENTION

The monitoring of pharmacokinetics, genetic, cellular, molecular orother types of events in vivo is of great interest to monitor drug orgene therapy efficacy as well as disease status or progression in smalllaboratory mammals and in the human body. In this respect, fluorescenceimaging, both in vitro and in vivo, has been used extensively togenerate anatomical and functional information from within cells andorganisms.

Fluorescence imaging of internal parts of animals (including humans) foranatomical or functional purposes often involves the injection of anextrinsic fluorophore, typically chemically coupled with anothermolecule, that distributes within the animal and accumulatespreferentially in cells and organs of interest. Images are then acquiredby detecting the fluorescence and mapping the signal relative to theanatomy of the animal. However, the excitation and emission spectra ofsuch extrinsic fluorophores often overlap with those of intrinsicfluorophores such that the fluorescence signal is a combination of thesignals from each fluorophore. Furthermore, such studies are oftenconducted using more than one extrinsic fluorophores which may haveoverlapping spectra. As a result, fluorescence images often containundesirable signals that obscure the signal from the fluorophore ofinterest.

Methods commonly used to attenuate or eliminate unwanted fluorescencesignals are based on spectral differences of the fluorescence emission,fluorescence lifetime differences (e.g. FLIM), or frequency domainhardware techniques. All of them have limitations. Methods based onspectral difference are limited to fluorophores having emission spectrathat do not significantly overlap thereby allowing acquisition offluorescence at a non-overlapping wavelength which is specific for aparticular fluorophore. Methods based on fluorescence lifetime helpdistinguish signals from different fluorophores but do not retain theinformation related to fluorophore intensity and consequentlyinformation related to concentration of the fluorophore is lost.Frequency domain hardware techniques require multiple image acquisitionat a plurality of phase delays to suppress unwanted fluorescence and aretherefore time consuming.

Accordingly, it would be desirable to be provided with a fluorescenceimaging method overcoming the above mentioned deficiencies.

SUMMARY OF THE INVENTION

The present invention provides an improved method for enhancing contrastand specificity of fluorescence images of an object, such as abiological tissue, by selectively eliminating or reducing unwantedfluorescence from fluorophores other than the fluorophore of interest.The method is based on the generation of intensity images weighted as afunction of measured lifetime in which the intensity information isconserved and hence information related to the concentration of thefluorophore of interest.

Thus in one embodiment there is provided a method for optical imaging ofan object containing two or more fluorophore species in which afluorescence signal is acquired, using time domain or frequency domain,for one or more region of interest (ROI) of the object using anexcitation and an emission wavelength compatible with detection of atleast one of the two or more fluorophore species. A fluorescenceintensity and a fluorescence lifetime are calculated from thefluorescence signals for each of the pixels and the fluorescenceintensity is multiplied by a weighting factor. The weighting factor is afunction of the calculated fluorescence lifetime and one or morepredetermined fluorescence lifetime of the fluorophore species and isused to generate a weighted fluorescence intensity for each pixel of theROI from which a weighted fluorescence intensity image can be obtained.

In a further embodiment, the method also provides for a adjustment ofthe fluorescence intensity to account for the relative contribution ofeach fluorophore. Thus when the fluorescence signal comprisescontribution from two or more fluorophore species a contributionfraction is derived for at least one of the fluorophore species and theweighted fluorescence intensity is multiplied by the contributionfraction. The contribution fraction can be determined, for example, byfitting a temporal point spread function (TPSF) of the fluorescencesignal with an exponential decay function.

In yet a further embodiment, the method provides for a primary weightingstep which can substantially reduce background fluorescence signal fromintrinsic fluorophore species. Thus the fluorescence intensity signalcan be multiplied by a primary weighting factor prior to the step ofmultiplying the fluorescence intensity by a weighting factor, theprimary weighting factor being a function of the calculated fluorescencelifetime and two predetermined fluorescence lifetimes of two or morefluorophore species that are being imaged.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features and advantages of the present invention will becomeapparent from the following detailed description, taken in combinationwith the appended drawings, in which:

FIG. 1A is a schematic representation of the generation of an intensityimage from fluorescence signal from a region of interest (ROI) of anobject;

FIG. 1B is a schematic representation of the generation of a lifetimeimage from fluorescence signal from a region of interest (ROI)of anobject;

FIG. 2 is a flow chart of an embodiment of the invention in which aweighted intensity image is obtained from a raw intensity image;

FIG. 3 is a flow chart of an embodiment of the invention in which acontribution fraction adjusted weighted intensity image is obtained froma raw intensity image;

FIG. 4 is a flow chart of an embodiment of the invention in which aprimary weighting is applied to the raw intensity image;

FIG. 5(a-i) is a raw fluorescence intensity (integration over time ofthe TPSF in each pixel) image of a [55%:45%] mixture of two fluorophorespecies namely Cy 5.5 and Atto 680;

FIG. 5(a-ii) is an effective lifetime image generated by fitting thefluorescence TPSF from the dual-dye mixture in each pixel with amono-exponential decay model.

FIG. 5(a-iii) exhibits a processed intensity image (I_(new)) obtained byperforming a preliminary weighting on the raw intensity image FIG.1(a-i);

FIG. 5(b-i) exhibits a fluorescence lifetime image of Cy5.5 calculatedby dual exponential fitting of the fluorescence TPSF of the dual-dyemixture in each pixel;

FIG. 5(b-ii) exhibits an intensity fraction image of Cy 5.5 calculatedby dual exponential fitting of the fluorescence TPSF of the dual-dyemixture in each pixel;

FIG. 5(b-iii) exhibits a weighted fluorescence intensity image (I¹ _(w))of Cy 5.5 obtained by the method of the invention; At each pixel, thefluorescence intensity is related to the concentration of Cy5.5 at thatlocation;

FIG. 5(c-i) exhibits a fluorescence lifetime image of Atto680 calculatedby dual exponential fitting of the fluorescence TPSF of the dual-dyemixture in each pixel;

FIG. 5(c-ii) exhibits an intensity fraction image of Atto680 calculatedby dual exponential fitting of the fluorescence TPSF of the dual-dyemixture in each pixel;

FIG. 5(c-iii) exhibits a weighted fluorescence intensity image (I² _(w))of Atto680 obtained by the method of the invention; At each pixel, thefluorescence intensity is related to the concentration of Atto680 atthat location;

FIG. 6(i) is a raw fluorescence intensity (integration over time of theTPSF in each pixel) image of Cy 5.5 and Atto680; on the left is Atto680;On the right is Cy5.5; One can not distinguish the fluorescence byfluorescence intensity only;

FIG. 6(ii) is a fluorescence lifetime image of the fluorophores Atto680and Cy5.5; One can distinguish the two fluorophores by theirfluorescence lifetime; This is the mechanism behind the fluorescencelifetime image; However, fluorescence intensity (and thus concentration)information is lost in this image;

FIG. 6(iii) is a fluorescence intensity image of Cy5.5 extracted fromFIG. 6(i) using the method of the invention; and

FIG. 6(iv) is a fluorescence intensity image of Atto 680 extracted fromFIG. 6(i) using the method of the invention.

It will be noted that throughout the appended drawings, like featuresare identified by like reference numerals.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention provides an improved method for enhancingfluorescence images of an object, such as a biological tissue, byselectively eliminating or reducing unwanted fluorescence fromfluorophores other than the fluorophore of interest. The method is basedon the measurement of the fluorescence intensity and lifetime offluorophores. The resulting image preserves information related to thefluorescence intensity (and thus the concentration) of the fluorophoreof interest. It will be appreciated that the method may be applied todifferent techniques such as optical imaging, time-resolved fluorescencemicroscopy and the like.

In the present disclosure by fluorophore species it is meantfluorophores having different fluorescence lifetime. Thus fluorophorespecies may refer to different fluorophore molecules but it may alsorefer to the same fluorophore molecule in different environments witheach environment conferring the fluorophore a different fluorescencelifetime. For example, conditions such as pH, viscosity, temperature andthe like are known to affect the lifetime of fluorophores. Theenvironment may also refer to the molecular environment of thefluorophore. For example a fluorophore that is free typically exhibits adifferent lifetime than the same fluorophore bound to another molecule.The term fluorophore may refer to small molecules or to macromoleculessuch as proteins that may comprise molecular electronic configurationscapable of emitting fluorescent light when excited.

In one embodiment of the present invention the lifetime of a fluorophorespecies and intensity of the fluorescence are obtained using time domain(TD) data acquisition. A time resolved fluorescence image can beobtained by exciting a fluorophore of interest with a pulsed lightsource at a fluorescence excitation wavelength and by collecting thefluorescence signal at a fluorescence emission wavelength using atime-resolved photo detector. The pulsed light source can be any type ofpulsed laser (e.g. diode laser, solid state laser, gas laser etc.) orother pulsed light sources (e.g. pulsed lamp). The time-resolved photodetector can be a photo multiplier tube (PMT), avalanche photodiodes(APD), PIN coupled with time correlated single photon counting (TCSPC),a streak camera, or a gated intensified charged coupled device (ICCD).

The fluorescence image can be generated by direct imaging of thefluorescent object using a camera or by raster scanning the fluorescentobject using a point detector and reconstructing the image usinginformation from each detection point (pixel). An example of the lattermodality is employed by the explore Optix™ imager described ininternational patent application WO 2004/044562 A1 which is incorporatedherein by reference.

While the embodiments of the invention will be described using timedomain as an exemplary modality of data acquisition, it will beappreciated that the method of the invention may also be applied usingfrequency domain data acquisition. In frequency domain, one can obtainfluorescence intensity and lifetime by measuring modulation of amplitudeand phase shift. Such measurements are well known in the art (Hawryszand Sevick-Muraca, Neoplasia vol. 2 (5), 2000 p. 388-417 which isincorporated herewith by reference).

As shown in FIG. 1A, each signal acquisition corresponds to a temporalpoint spread function (TPSF) 10 of the fluorescence signal emitted bythe object at a specific detection point 12. By time integrating, eithercompletely or partially, the TPSF at each pixel, one can get afluorescence intensity image 14, denoted by I_(ij) which can provideinformation on the concentration and the localization of thefluorophores. Furthermore by mathematically fitting the TPSF, one canobtain the fluorescence lifetime which can be used to generate alifetime image 18, denoted by τ_(ij) (FIG. 1B). If the fluorescencesignal is from more than one fluorophore species, a multi-exponentialdecay model can be used to fit the TPSF and to derive lifetimes for eachfluorophore species and contribution fractions of the species.

In most practical cases a TPSF measured at a given point is usuallycomposed of several TPSFs due to the various endogenous and exogenousfluorophore species present in the system. The measured TPSF may then bewritten as, $\begin{matrix}{{TPSF}_{meas} = {\sum\limits_{k = 1}^{n}\quad{f_{ij}^{k} \times {TPSF}_{k}}}} & (1)\end{matrix}$

where TPSF_(k) is the fluorescence TPSF of k^(th) component (fluorophorespecies) considered in the n-component analysis, and f^(k) is itscontribution fraction. Generally, as mentioned above, curve fittingmethods are required to resolve the measured TPSF into its componentconstituents. By convolving the system Impulse Response Function (IRF)with the modeled TPSFs of the components, an approximation of themeasured TPSF may be made. With the use of numerical curve fittingmethods, estimates of the lifetimes and/or relative fractionalcontribution of each of the n components can be obtained.

For example, it is possible to model the contributions of differentfluorophore species with the following expression (example provided fortwo fluorophore species):f¹ _(ij) exp(−t/τ ₁)+f ² _(ij) exp(−t/τ ₂)   (2)with f_(ij) ¹+f_(ij) ²=1

where t is the time, and f_(ij) ¹ and f_(ij) ² are contributionfractions (amplitude factors) and exp(−t/τ₁) and exp(−t/τ₂) model thefalling slope of each of the two individual TPSFs. The amplitude factorsare related to the concentrations of the species and their fluorescentefficiencies (e.g. quantum yields, extinction coefficients, excitationlaser wavelength, fluorescence spectrum, filter window, etc.).

Referring to FIG. 2 and assuming that the fluorophore species ofinterest (i.e. from which the image is to be reconstructed) has afluorescence lifetime τ, the intensity image 14 can be multiplied at 20by a weighting factor which is a function of the fluorescence lifetime τand the measured (effective) fluorescence lifetime τ_(ij), and used togenerate a weighted fluorescence intensity image at 22 which isrepresentative of the distribution and the concentration of thefluorophore species of interest.

In one embodiment, the weighting factor is determined by an Indicator(or Rect) function defined by a range of user determined lifetimes thatencompasses the measured lifetime τ_(ij) at a particular pixel. Pixelsexhibiting lifetimes outside the predetermined range can be weightedaccordingly or simply eliminated.

In a preferred embodiment the error Δτ derived from the fitting of theTPSF to calculate the effective lifetime can be used to determine therange. Thus one can generate a logical image map L_(ij) by the followingcriteria: $\begin{matrix}{L_{ij} = \left\{ \begin{matrix}1 & {if} & {{\tau - {\Delta\tau}} < \tau_{ij} < {\tau + {\Delta\tau}}} \\0 & \quad & {otherwise}\end{matrix} \right.} & (3)\end{matrix}$

By element-wise multiplying this matrix to the raw intensity imageI_(ij), one could get a weighted intensity image I_(w), in whichunwanted fluorescence and/or noise are suppressed. It will be noted thatthis treatment of the fluorescence signal retains the intensityinformation of the fluorescence signal.

In the case where two (or more) fluorophore species are contributing tothe TPSF, one can obtain the fluorescence lifetimes τ_(ij) ¹ and τ_(ij)² (more generally τ^(n) _(ij), and the contribution fractions ƒ_(ij) ¹and ƒ_(ij) ² (more generally ƒ_(ij) ^(n)) where f_(ij) ¹+f_(ij) ²=1 ofthe two fluorophores by fitting the TPSF by using a dual exponentialdecay model.

Referring now to FIG. 3, by element-wise multiplying matrix ƒ_(ij) ^(n)to I_(w) at 30, one can get a new intensity image I^(n) _(w) at 32,which is proportional to the intensity of fluorophore species n. If only2 fluorophore species are present image I² _(w) can be obtained by themethod summarized in FIG. 3 or by simply subtracting I¹ _(w) from I_(w).It will be appreciated that the contribution fractions can be multipliedto the raw intensity image I before performing the weighting step.

Referring now to FIG. 4, it may be advantageous to “clean” the intensityimage, prior to weighting, by performing a preliminary weighting at 40based on the lifetimes of at least two fluorophore species. For example,in an object comprising two fluorophore species with fluorescencelifetimes τ₁ and τ₂, and where τ₁<τ₂, then the measured fluorescencelifetime τ satisfies τ₁<τ<τ₂ if a single exponential decay model isused. Further assuming the fitting error is Δτ, then one can generate alogical image map by the following criteria $\begin{matrix}{L_{ij} = \left\{ \begin{matrix}1 & {if} & {{\tau_{1} - {\Delta\tau}} < \tau_{ij} < {\tau_{2} + {\Delta\tau}}} \\0 & \quad & {otherwise}\end{matrix} \right.} & (4)\end{matrix}$

By element-wise multiplying this matrix to the intensity image I_(ij),one can generate a new intensity image I_(new) at 42 which is backgroundsuppressed. The background may comprise, for example, fluorescence fromintrinsic molecules. I_(new) can then be used in the process describedin FIGS. 2 and 3 to obtain I_(w), I¹ _(w), etc.

It will be appreciated that the method described above can be extendedto multi-fluorophore species using a multi-exponential decay model forfluorescence lifetime fitting instead of dual exponential decay model.

It will also be appreciated that the ranges of lifetime on which theweighting is based can be defined by the user according to the desiredfluorescence information. In a preferred embodiment, the ranges aredefined by the expected (τ^(n)) lifetime of the fluorophore species.

For applications such as diagnosis and pharmacological studies, it isoften desirable to have an image that provides information on theconcentration and depth of the fluorophore species. However, to assumethat the fluorescence intensity signal is proportional to the flurophoreconcentration can be misleading since the depth of the flurophore willalso impact the fluorescence intensity signal. Thus to generate an imagethat reflects the concentration of the fluorophore species thepropagation loss of the fluorescence due to tissue absorption andscattering should be taken in consideration. An example of concentrationdetermination is provided below.

EXAMPLES Example 1

Equal volumes of 50 nM Cy5.5 and 150 nM Atto680 were mixed together.Fluorescence signal was obtained using eXplore Optix™ with a pulseddiode laser wavelength at 666 nm as the excitation light source. Whenthe quantum yield, extinction coefficient, and fluorescence spectrum andfilter window information are taken into account, the fluorescencesignal ratio of Cy5.5 and Atto680 from the mixture is about 0.55:0.45.

FIG. 5 illustrates the method described above. Panel(a-i) is a rawfluorescence intensity image of the Cy 5.5 and Atto 680 mixture. Alifetime image (panel (a-ii)) was generated using an effective lifetime(fitting the TPSF with a single exponential). Panel (a-iii) exhibits aprocessed intensity image (I_(new)) obtained by performing a preliminaryweighting on the raw intensity image. Because only Cy5.5 and Atto 680are present there is no difference between the raw image and processedimage (no background fluorescence). Panels(b-i) and (c-i) exhibitlifetime images based on the lifetime of one fluorophore species onlyafter dual exponential fitting of the TPSF. Panels (b-ii) and (c-ii)exhibit a contribution factor image of the fluorophore species. Bothlifetime and fraction are obtained at the same time by direct fitting ofthe TPSF in each pixel using a dual exponential decay model. Panels(b-iii) and (c-iii) exhibit a weighted fluorescence intensity image (I¹_(w),I² _(w)) obtained by the method described above. In the presentexample the fluorescence intensity is proportional to the concentrationof Cy5.5 or Atto680 since both fluorophore are at the same depth(phantom surface);

Example 2

One hundred nM Cy5.5 and 200 nM Atto680 solution were arranged in twoseparate locations. Fluorescence signal was obtained using eXplore Optixwith a pulsed diode laser wavelength at 666 nm as the excitation lightsource.

In the particular case where the location of fluorophore species withinthe object are not overlapping, there is no need for multi-exponentialfitting of the TPSF and one can proceed directly with the weighting stepof the method. FIG. 6 provides such an example in which the twofluorophores species do not overlap. Panel (i) of FIG. 6 is a measuredraw fluorescence intensity image with two fluorophore species, Atto 680on the left and Cy5.5 on the right. From the intensity image alone,without knowing a priori where the fluorophores are located, one wouldnot be able to identify the fluorophores species. Panel (ii) is thecorresponding fluorescence lifetime image obtained by fitting the TPSFof each pixel with a single exponential decay model. While the lifetimeimage enables the determination of the species of the fluorophore if thelifetimes are known a priori, it does not convey any intensityinformation. However, when the method of the present invention is usedthe intensity information is preserved. Thus in the example providedbelow the weighting function was based on a range of lifetimesdetermined to be between 0.9 and 1.05 ns for Cy 5.5 and 1.7 and 1.83 nsfor Atto 680. using the criteria: $L_{ij} = \left\{ \begin{matrix}1 & {if} & {0.9 < \tau_{ij} < 1.05} \\0 & \quad & {otherwise}\end{matrix} \right.$for Cy 5.5 one obtains the image displayed in panel (iii) and$L_{ij} = \left\{ \begin{matrix}1 & {if} & {1.7 < \tau_{ij} < 1.83} \\0 & \quad & {otherwise}\end{matrix} \right.$

for Atto 680 one obtains the image displayed in panel (iv). Both imagesretain the intensity information for the fluorophore of interest. Sincethey are both at the same depth (phantom surface), the intensity isrelated to their concentration through quantum yield, extinctioncoefficient, fluorescent spectrum and filter window.

Example 3

The fluorophore species may be the same fluorophore molecule indifferent environment. Thus, for example, the object may comprise onefluorophore having a lifetime τ₁ when it is bound to a protein and alifetime τ₂ when it is free. In this case it is possible to model theTPSF by the following dual exponential:f exp(−t/τ ₁)+(1−f)exp(−t/τ ₂)   (5)

where, t is the time, τ₁ and τ₂ are the respective lifetimes of thebound and free states and f is the fraction of fluorophores in the boundstate: f=[bound]/([bound]+[free]). The parameters in this model can thenbe obtained from measured data through multi-variate curve fitting. Thedual exponential for free/bound fluorophore species can be used toobtain weighted intensity images as described above.

Example 4

Under certain assumptions such as assuming that the optical propertiesof the medium are the same at the excitation and emission wavelength,the fluorescence intensity as a function of time can be expressed by theBorn approximation: $\begin{matrix}\begin{matrix}{{\phi\quad(t)} \cong {\sum\limits_{dipoles}\quad{\left( {{QC}\frac{r_{sp} + r_{pd}}{4\pi\quad{Dr}_{sp}r_{pd}}v\quad\left( {4\pi\quad{Dvt}} \right)^{{- 3}/2}{\mathbb{e}}^{{- \frac{{({r_{sp} + r_{pd}})}^{2}}{4\quad{Dvt}}} - {\mu_{a}{vt}}}} \right)*}}} \\{\left( \frac{{\mathbb{e}}^{- \frac{t}{\tau}}}{\tau} \right)*({IRF})}\end{matrix} & (6)\end{matrix}$

Where:

r_(ap) is the distance from source s (point on the object at which lightis injected) to fluorophore depth position P;

r_(pd) is the distance from fluorophore depth position p to detector d;

μ_(a) is the optical absorption coefficient;

D is the optical diffusion coefficient,$D = \frac{1}{3\quad\mu_{s}^{\prime}}$where; μ_(s)′ is the reduced optical scatter coefficient;

ν is the speed of light in the medium;

Q is the quantum efficiency;

C is the concentration of the fluorophore;

τ is the lifetime of the fluorophore;

the symbol * refers to the operation of convolution and

IRF is the impulse response function of the instrument used to measurefluorescence.

By setting the first derivative of equation 6 as a function of timeequal to zero, the time position of the maximum of the TPSF (t_(max))can be found. Under certain approximations (absorption is small at timeshorter than t_(max), the scatter coefficient is known or can beapproximated) and by assuming that r_(sp) is approximately equal tor_(pd), it is found that the following equation can be derived fromequation 6: $\begin{matrix}{t_{\max} \cong \frac{d\sqrt{\tau}}{\sqrt{Dv}}} & (7)\end{matrix}$

where d is the depth of the fluorophore object.

For a given depth, the intensity I of the emission signal detected atthe surface can be related to fluorophore concentration by the opticalproperties of the medium (absorption and scattering coefficients) andthe depth of the fluorophore. $\begin{matrix}{I \propto {C\quad{\mathbb{e}}^{- \sqrt{\frac{\mu_{a}}{D}d}}}} & (8)\end{matrix}$

Using time-domain information as described above, the depth d can bedetermined. Isolating C in equation 8 and knowing signal intensity anddepth of the fluorophore, one can thus recover the concentration offluorophore (i.e. the amount of fluorescent molecules per unit volume)within an accuracy that depends exponentially on the recovered depthaccuracy. Thus, in another aspect of the invention, estimates of therelative concentration of the fluorophore, Conc._(Relative), can beobtained by determining its depth, d, and normalizing the surfaceintensity measurement, I, as follows (Equation 9):Conc._(Relative) =Id ² e ^(2d√{square root over (μn/D)})  (9)

under certain assumptions, equation 9 can be derived from equation 1.

If the fluorophore objects are not at the surface of the tissue, themethod described above can be used to obtain their concentration mapfrom the weighted intensity image.

1. A method for optical imaging of an object containing two or morefluorophore species, said method comprising: acquiring a fluorescencesignal comprising time and amplitude information for one or more pixelof a region of interest (ROI) of said object using an excitation and anemission wavelength compatible with detection of at least one of saidtwo or more fluorophore species; calculating a fluorescence intensityand a fluorescence lifetime from said fluorescence signal for each ofsaid one or more pixel; multiplying said fluorescence intensity by aweighting factor, said weighting factor being a function of saidcalculated fluorescence lifetime and at least one predeterminedfluorescence lifetime, to generate a weighted fluorescence intensity forsaid one or more pixel; generating an image of said ROI based on saidweighted fluorescence intensity of said one or more pixel.
 2. The methodas claimed in claim 1 wherein said fluorescence signal comprisescontribution from two or more fluorophore species, the method furthercomprising steps of: deriving a contribution fraction for at least oneof said fluorophore species; and multiplying said weighted fluorescenceintensity by said contribution fraction.
 3. The method as claimed inclaim 2 wherein said contribution fraction is determined by modelingsaid fluorescence signal with a multi-exponential function.
 4. Themethod as claimed in claim 3 wherein said modeling is applied to atemporal point spread function (TPSF) of said fluorescence signal. 5.The method as claimed in claim 1 wherein said weighting factor isdetermined by an Indicator function.
 6. The method as claimed in claim 5wherein said Indicator function is defined by boundaries which arefunction of said predetermined fluorescence lifetime.
 7. The method asclaimed in claim 6 wherein said boundaries are also function of an errorassociated with said measured lifetime.
 8. The method as claimed inclaim 7 wherein said weighting factor is 1 when said measured lifetimeis within said boundaries and 0 otherwise.
 9. The method as claimed inclaim 2 further comprising a step of: multiplying said fluorescenceintensity by a preliminary weighting factor prior to said step ofmultiplying said fluorescence intensity by said weighting factor or saidcontribution fraction, said preliminary weighting factor being afunction of said calculated fluorescence lifetime and two predeterminedfluorescence lifetimes corresponding to expected lifetimes of said twoor more fluorophore species.
 10. The method as claimed in any one ofclaim 1-9 wherein said fluorescence signal acquisition is selected fromfrequency domain and time domain modality.
 11. The method as claimed inany one of claim 1-9 wherein said fluorescence species comprises afluorophore that is distributed between a free state and a bound state.12. The method as claimed in any one of claim 1-9 wherein said weightedintensity is further processed to yield concentration of at least one ofsaid two or more fluorophore species.
 13. A method for optical imagingof an object containing two or more fluorophore species, said methodcomprising: acquiring a fluorescence signal, said signal comprisingfluorescence from said two or more fluorophore species and comprisinglifetime information for one or more pixel of a region of interest (ROI)of said object; calculating a fluorescence intensity and a fluorescencelifetime from said fluorescence signal for each of said one or morepixel; deriving a contribution fraction for at least one of saidfluorophore species; multiplying said calculated fluorescence intensityby said contribution fraction to generate a species weightedfluorescence intensity; multiplying said species weighted fluorescenceintensity by a weighting factor, said weighting factor being a functionof said calculated fluorescence lifetime and at least one predeterminedfluorescence lifetime, to generate a weighted fluorescence intensity forsaid one or more pixel; generating an image of said ROI based on saidweighted fluorescence intensity of said one or more pixel.
 14. Themethod as claimed in claim 13 wherein said contribution fraction isdetermined by modeling said fluorescence signal with a multi-exponentialfunction.
 15. The method as claimed in claim 14 wherein said modeling isapplied to a temporal point spread function (TPSF) of said fluorescencesignal.
 16. The method as claimed in claim 13 wherein said weightingfactor is determined by an Indicator function.
 17. The method as claimedin claim 16 wherein said Indicator function is defined by boundarieswhich are function of said predetermined fluorescence lifetime.
 18. Themethod as claimed in claim 17 wherein said boundaries are also functionof an error associated with said measured lifetime.
 19. The method asclaimed in claim 18 wherein said weighting factor is 1 when saidmeasured lifetime is within said boundaries and 0 otherwise.
 20. Themethod as claimed in claim 13 further comprising a step of: multiplyingsaid fluorescence intensity by a preliminary weighting factor prior tosaid step of multiplying said fluorescence intensity by saidcontribution fraction, said preliminary weighting factor being afunction of said calculated fluorescence lifetime and two predeterminedfluorescence lifetimes corresponding to expected lifetimes of said twoor more fluorophore species.
 21. The method as claimed in any one ofclaim 13-20 wherein said fluorescence signal acquisition is selectedfrom frequency domain and time domain modality.
 22. The method asclaimed in claim 13-21 wherein said fluorescence species comprises afluorophore that is distributed between a free state and a bound state.23. The method as claimed in claim 13-21 wherein said weighted intensityis further processed to yield concentration of at least one of said twoor more fluorophore species.